This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The distribution of oxygen within animal cells and how this interfaces with the hypoxia response machinery, especially the 'oxygen sensing'hypoxia inducible factor hydroxylases, is a major and biomedicinally important question in hypoxia biology. In collaboration with the Oxford-based Ratcliffe and Pugh laboratories (Clinical medicine) Professor Schofield's group studies the Molecular Mechanism of the Hypoxic Response using techniques such as proteomics, X-ray crystallography, biological mass spectrometry, molecular biology, kinetics and organic synthesis. Here we will use the ESR Microscopy facility at ACERT providing micrometer resolution of investigated tissues and cells to analyze oxygen distributions in different cell types under oxygen tensions ranging from hypoxia (2%) to normoxia. We will correlate these analyses with the activities of the sensing enzymes via a combined cell-biology and EPR led biophysical approaches. The cells will include those with mutant versions of the oxygen sensing enzymes that have varied kinetic properties with respect to oxygen binding and reaction.